Head and neck squamous cell carcinoma (HNSCC) is the 6th most common type of cancer. Survival rates for advanced disease are poor, and treatment leads to impairment in speech, hearing, swallowing, and quality of life. The goal of our laboratory is to improve upon treatments for HNSCC by increasing their efficacy and decreasing toxicity. Standard treatments including cisplatin and other platinum-based chemotherapy drugs and radiation. Immune checkpoint inhibitors have also recently been FDA approved for the treatment of HNSCC. One major goal of the lab is to determine how cisplatin chemotherapy affects the immune system, in order to best design treatment paradigms that include new immunotherapy drugs or novel agents targeting specific mutations in HNSCC. Other goals include characterizing the toxicities of cisplatin chemotherapy in HNSCC patients, with a focus on characterizing and preventing cisplatin-induced hearing loss. Preclinical studies during this year in the lab have established that cisplatin alters specific aspects of anti-tumor immunity, including antigen processing and presentation. In this process, mutated peptides in the tumor cells are presented to immune cells including antigen presenting cells and T cells, which then kill the tumor cells. The processing of peptides for presentation as antigens involves a network of chaperones in the endoplasmic reticulum, which help to load the antigenic peptides onto major histocompatibility complex (MHC) molecules for presentation to immune cells. We have shown in several human HNSCC cell lines that levels of MHC class I and several other chaperones increase within the tumor cells following treatment with cisplatin, which is a favorable effect of cisplatin on anti-tumor immunity. In addition, cisplatin alters the expression of programmed death ligand 1 (PD-L1) on tumor cells. This immune checkpoint ligand interacts with the PD-1 receptor on T cells, inhibiting their anti-tumor immune function. We have found in HNSCC cell lines and in an in vivo mouse model of HNSCC that treatment with cisplatin increases the expression of PD-L1 on tumor cells. The increase in PD-L1 is a potentially unfavorable effect of cisplatin on anti-tumor immunity, but also provides a rationale for using a drug to block the PD-L1/PD-1 pathway in combination with cisplatin for HNSCC. Experiments using a syngeneic mouse model of HNSCC with a fully intact immune system showed that cisplatin and a PD-L1-blocking antibody have synergistic anti-tumor activity. This work has been submitted for publication. Another project has established that cisplatin chemotherapy also induces a process called immunogenic cell death, whereby cells upon treatment release damage signals and activate anti-tumor immunity. We are in the process of completing the final experiments for submission of a manuscript. A third project investigated the efficacy of a novel targeted anti-cancer drug, the IAP inhibitor ASTX660, in combination with cisplatin chemotherapy, radiation, or immunotherapy in our preclinical mouse model of HNSCC. ASTX660 demonstrated additive or synergistic anti-tumor activity with all of these standard agents. This project has been submitted for publication. Another focus is the characterization and prevention of hearing loss in HNSCC patients treated with cisplatin. Cisplatin is known to cause permanent damage to mechanosensory hair cells and other structures of the inner ear, causing high-frequency hearing loss. Patients with HNSCC are at particular risk, since many are treated with cisplatin as well as radiation near the inner ear. Intramural collaborations have been established within NIDCD to investigate the incidence and severity of cisplatin-induced hearing loss in HNSCC patients treated with low-dose weekly cisplatin combined with radiation. A clinical trial to achieve this at NIH has been IRB approved. Another preclinical collaborative project examined whether mice treated with cisplatin were at increased risk with for hearing loss or kidney toxicity with the addition of anti-PD-1 immunotherapy, which has recently been FDA approved for treatment of HNSCC. Results indicate that immunotherapy does not exacerbate cisplatin-induced toxicity, and we are preparing these data for publication.